DNA molecules stabilized by modifications of the 3′-terminal phosphodiester linkage

ABSTRACT

The use of oligodeoxynucleotides modified at the 3′-terminal internucleotide link as therapeutic agents by a method of hybridizing the modified oligonucleotide to a complementary sequence within a targeted mRNA and cleaving the mRNA within the RNA-DNA helix by the enzyme RNaseH to block the expression of the corresponding gene.

This is a division of application Ser. No. 07/672,088 filed Mar. 19,1991, now U.S. Pat. No. 5,491,133, which is a continuation ofapplication Ser. No. 07/126,564 filed on Nov. 30, 1987, now abandoned.

GRANT REFERENCE

The invention described herein was made in part in the course of workunder grants from the National Institutes of Health, Grant Nos. HL-33555and AM-25295.

BACKGROUND OF THE INVENTION

It is well known that nucleic acids, i.e., deoxyribonucleic acids (DNA)and ribonucleic acids (RNA) are essential building blocks in livingcells. These compounds are high molecular weight polymers which are madeup of many “nucleotide” units, each such nucleotide unit being composedof a base (a purine or a pyrimidine), a sugar (which is either ribose ordeoxyribose) and a molecule of phosphoric acid. DNA contains deoxyriboseas the sugar moiety and the bases adenine, guanine, cytosine, andthymine (which may be represented as A, G, C and T, respectively). RNAcontains ribose instead of deoxyribose and uracil (U) in place ofthymine.

The nucleotide units in DNA and RNA are assembled in definite linearsequences which determine specific biological functions. In the normalmammalian cell, DNA replicates itself, and also serves as the templatefor the synthesis of RNA molecules whose nucleotide sequences carry theinformation encoded by the DNA. RNA molecules serve several differentfunctions within the cell. Messenger RNA (mRNA) directs proteinsynthesis.

According to the well known Watson-Crick model, DNA molecules consist oftwo polynucleotide strands coiled about a common axis. The resultingdouble helix is held together by hydrogen bonds between complementarybase pairs in each strand. Hydrogen bonds are formed only betweenadenine (A) and thymine (T) and between guanine (G) and cytosine (C).Hence within the double helix, adenine (A) and thymine (T) are viewed ascomplementary bases which bind to each other as A-T. The same is truefor guanine (G) and cytosine (C), as G-C.

In a single polynucleotide strand, any sequence of nucleotides ispossible. However, once the order of bases within one strand of a DNAmolecule is specified, the exact sequence of the other strand issimultaneously determined due to the indicated base pairing.Accordingly, each strand of a DNA molecule is the complement of theother. In the process of DNA replication, the two strands act astemplates for the synthesis of two new chains with complementarynucleotide sequences. The net result is the production of two new DNAmolecules each containing one of the original strands plus a newlysynthesized strand that is complementary to it. This process is referredto as semiconservative replication and allows the genetic informationencoded within the DNA, specified by the nucleotide sequence, to betransmitted from one generation to the next.

Collectively, the genetic information of an organism is termed thegenome. The genome of bacteria and of all higher species is composed ofDNA. The genome of viruses may be either DNA or RNA. In any case, thegenome of any particular species, whether a virus, bacteria, or higherorganism has a characteristic nucleotide sequence which, stated simply,can be viewed as the “fingerprint” of that species.

In the process of transcription, DNA is copied, or transcribed, intoRNA. The RNA molecule that is synthesized is single stranded. Itsnucleotide sequence is complementary to a segment of one of the twostrands of the DNA molecule, and hence it is an exact copy of theopposite strand except that thymine is replaced with uracil. The lengthof DNA that is copied to form a single RNA molecule represents one gene.The human genome contains approximately 50,000 genes.

Messenger RNAs (mRNAs) carry the coding information necessary forprotein synthesis. The sequence of nucleotides in the mRNA moleculespecifies the arrangement of amino acids in the protein whose synthesiswill be directed by that mRNA. Each set of three nucleotides, a unitcalled a codon, specifies a particular amino acid. The process by whichmRNAs direct protein synthesis is termed translation. Translation ofmRNAs occurs on ribosomes within the cytoplasm of the cell.

The flow of information from gene to protein can thus be represented bythe following scheme:

For each protein made by an organism there exists a corresponding gene.

It is generally known in the field of molecular biology thatoligodeoxyribonucleotides, short single-stranded DNA fragments, having anucleotide sequence complementary to a portion of a specified mRNA maybe used to block the expression of the corresponding gene. This occursby hybridization (binding) of the oligonucleotide to the mRNA, accordingto the rules of base pairing described above, which then preventstranslation of the mRNA. It has now been discovered that the inhibitionof translation observed is due to cleavage of the mRNA by the enzymeRNaseH at the site of the RNA-DNA double helix formed with theoligonucleotide (see FIG. 1). Hybridization of the oligonucleotide tothe mRNA is generally not sufficient in itself to significantly inhibittranslation. Evidently an oligonucleotide bound to mRNA can be strippedoff in the process of translation. An important corollary of this resultis that for any modified oligonucleotide to efficiently block expressionof a gene, the hybrid formed with the selected mRNA must be recognizedby RNaseH and the mRNA cleaved by the enzyme.

The process of hybrid-arrested translation outlined in FIG. 1 hasobvious implications for the use of oligonucleotides as therapeuticagents. By selectively blocking the expression of a particular geneessential for the replication of a certain virus or bacteria anoligonucleotide could serve as an antimicrobial agent. Similarly, anoligonucleotide targeted against a gene responsible or required for theuncontrolled proliferation of a cancer cell would be useful as ananticancer agent. There are also applications in the treatment ofgenetic diseases such as sickle cell disease and thalassemia for whichno adequate treatment currently exists. Any gene can be targeted by thisapproach. The problem of drug specificity, a major hurdle in thedevelopment of conventional chemotherapeutic agents, is immediatelysolved by choosing the appropriate oligonucleotide sequencecomplementary to the selected mRNA. This circumvents toxicity resultingfrom a lack of selectivity of the drug, a serious limitation of allexisting antiviral and anticancer agents.

In addition to the use of oligonucleotides to block the expression ofselected genes for the treatment of diseases in man, other applicationsare also recognized. Additional applications include but are not limitedto the use of such oligonucleotides in veterinary medicine, aspesticides or fungicides, and in industrial or agricultural processes inwhich it is desirable to inhibit the expression of a particular gene byan organism utilized in that process.

A major problem limiting the utility of oligonucleotides as therapeuticagents is the rapid degradation of the oligonucleotide in blood andwithin cells. Enzymes which degrade DNA or RNA are termed nucleases.Such enzymes hydrolyze the phosphodiester bonds joining the nucleotideswithin a DNA or RNA chain, thereby cleaving the molecule into smallerfragments.

In the past there has been some progress made in the development ofoligonucleotide analogs that are resistant to nuclease degradation, butthe use of such derivatives to block the expression of specificallytargeted genes has met with limited success. See, for example, Ts'o etal. U.S. Pat. No. 4,469,863 issued Sep. 4, 1984; Miller et al. U.S. Pat.No. 4,507,433 issued Mar. 26, 1985; and Miller et al. U.S. Pat. No.4,511,713 issued Apr. 16, 1985. Each of the above patents have in commonthe objective of blocking the expression of selected genes, but theapproach taken has been less than successful judging from the highconcentrations of oligonucleotide required and a corresponding lack ofselectivity. The work described in each of the three prior patentsmentioned involves the use of oligonucleotides in which all of thephosphate groups have been modified in the form of methylphosphonates:

B, and B₂ are nucleic acid bases (either A, G, C or T).

In particular, U.S. Pat. No. 4,469,863 involves inter alia,methylphosphonate modification of oligonucleotides. U.S. Pat. No.4,507,433 involves a process for synthesizing deoxyribonucleotidemethylphosphonates on polystyrene supports; and U.S. Pat. No. 4,511,713involves determining the base sequence of a nucleic acid and hybridizingto it an appropriately synthesized oligonucleotide methylphosphonate tointerfere with its function.

The low level of activity observed using these fully modifiedmethylphosphonate analogs led us to suspect that they would not formeffective substrates for RNaseH when hybridized with mRNAs. Resultspresented in Example 2 show this to be the case.

In an effort to design new oligonucleotide analogs of greater use astherapeutic agents, we undertook a study of the pathways by whicholigonucleotides are degraded within blood and within cells. We havediscovered that the sole pathway of degradation in blood (Example 3) andthe predominant pathway in cells (Example 4) is via sequentialdegradation from the 3′-end to the 5′-end of the DNA chain in which onenucleotide is removed at a time. This pathway of degradation isillustrated in FIG. 2. These results provided for the first time arational basis for the design of oligonucleotide analogs modified so asto inhibit their degradation without interfering with their ability toform substrates for RNaseH.

It has now been discovered that oligonucleotides modified at only the3′-most internucleotide link are markedly protected from degradationwithin blood and within cells (Examples 5 and 6). Moreover, we havefound that such derivatives have normal hybridization properties and doform substrates with mRNAs that are recognized and cleaved by RNaseH,thereby preventing expression of the targeted gene (Example 7).

The accomplishment of the inhibition of expression of selected genes byoligonucleotides that are resistant to degradation, and that are,therefore, more effective when used therapeutically, is the primaryobjective of this invention.

Other objectives of the present invention include the preparation ofoligonucleotides modified at the 3′-phosphodiester linkage and theformulation of the modified oligonucleotide in a manner suitable fortherapeutic use. It is also apparent that DNA or RNA molecules somodified would be useful as hybridization probes for diagnosticapplications, in which case, the modification of the 3′-internucleotidelink would inhibit degradation of the probe by nucleases which may bepresent within the test sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration to show how the expression of aselected gene is blocked in accordance with the process of thisinvention by hybridization of an oligonucleotide to its complementarysequence within the mRNA and cleavage of the mRNA by the enzyme RNaseHat the site of the RNA-DNA double helix.

FIG. 2 illustrates the degradation of a single stranded DNA molecule(SEQ ID NO:5) by the action of a nuclease that removes sequentially onenucleotide at a time from the 3′- to the 5′-end of the chain.

FIG. 3 is a drawing of an autoradiogram demonstrating that the exclusivepathway of degradation of DNA fragments in human blood is byexonucleolytic cleavage from the 3′- to the 5′-end of the DNA chain. Theoligonucleotide utilized in this experiment, MA-15, has the sequence:5′-GAGCACCATGGTTTC (SEQ ID NO:1). In lanes 1-5, the oligonucleotide wasradiolabeled at the 5′-end of the molecule with ³²P. In lanes 6-10, the³²P-label is at the 3′-terminal internucleotide link. The labeledoligonucleotide was incubated in human blood at 37° C. Aliquots wereremoved at the times shown and analyzed by polyacrylamide gelelectrophoresis. The labeled bands within the gel were visualized byautoradiography.

FIG. 4 is a drawing of an autoradiogram demonstrating that anoligonucleotide in which only the 3′-most internucleotide link ismodified is completely resistant to degradation in human blood. Theoligonucleotide is a derivative of MA-15 in which the 3′-terminalinternucleotide link is a 2,2,2-trichloro-1,1-dimethylethylphosphotriester. Lane 1 is the oligonucleotide prior to incubation.Lanes 2-5 are aliquots taken after incubation of the oligonucleotide inblood for 15 minutes, 1 hour, 4 hours, and 18 hours, respectively. Thedecrease in intensity of the original band is due simply to removal ofthe 5′-end label rather than degradation of the oligonucleotide. Pi isinorganic phosphate.

FIG. 5 is a drawing of an autoradiogram of a Northern blot demonstratingthat oligonucleotides modified at the 3′-internucleotide link formsubstrates with mRNAs that are acted upon by RNaseH resulting incleavage of the mRNA at the site of the RNA-DNA double helix. Lane 1 ismouse globin mRNA alone. Lane 2 is mouse globin mRNA incubated withRNaseH in the absence of oligonucleotide. Lane 3 is globin mRNAhybridized with MA-15 and cleaved with RNaseH. In lane 4, the substratefor RNaseH is formed with the derivative of MA-15 in which the3′-terminal internucleotide link is a trichlorodimethylethylphosphotriester. In lane 5, the oligonucleotide used was furthermodified with naphthylisocyanate at the 5′-OH group of the molecule.Samples were electrophoresed on a 1.5% agarose gel. Afterelectrophoresis the gel was blotted onto Gene Screen Plus (DuPont), andbands were localized using ³²P labeled MA-15 as a probe for α-globinmRNA.

SUMMARY OF THE INVENTION

The invention comprises the method, means and composition which togetherenable the use of oligonucleotides that are modified at the 3′-terminalphosphodiester linkage, and are thereby rendered resistant todegradation within cells and body fluids, to selectively block theexpression of a particular gene. The method involves the hybridizationof the modified oligonucleotide to the corresponding mRNA to form asubstrate fully capable of being recognized by the enzyme RNaseH,followed by the cleavage of the mRNA at the site of the RNA-DNA doublehelix such that the expression of the targeted gene is blocked. Theinvention further details the use of DNA and RNA molecules modified atthe 3′-terminal phosphodiester linkage as nucleic acid probes fordiagnostic applications.

DETAILED DESCRIPTION OF THE INVENTION

In the broadest terms, the objectives of the present invention areaccomplished by modifying oligonucleotides at just the 3′-terminalphosphodiester linkage. When this is accomplished it has been found thatthe oligonucleotide is markedly resistant to degradation by nucleaseswithin cells and within blood. Equally important, such modifiedoligonucleotides hybridize normally with complementary nucleic acidsequences and do form substrates with mRNAs that are recognized andcleaved by RNaseH. As a result of the cleave of the mRNA, expression ofthe corresponding gene is selectively blocked as is desired in varioustherapeutic applications.

Recent advances in molecular biology employing recombinant DNAtechniques have led to new diagnostic and therapeutic strategies. In DNAdiagnostics, a DNA or RNA probe is used to detect the presence of acomplementary nucleic acid (DNA or RNA) sequence. In this process theprobe hybridizes to its complementary sequence if it is present withinthe sample. The target sequence may be a DNA or RNA molecule of abacteria or virus, or an altered gene leading to a genetic abnormalitysuch as sickle cell disease. Frequently, the sample to be analyzed isimmobilized on a solid support such as nitrocellulose or a nylonmembrane. The probe carries a label, e.g., a radioactive, fluorescent,or enzyme marker to permit its detection. The field of DNA diagnosticsis currently under very active investigation, having applications in thediagnosis of infectious diseases, cancer, and genetic disorders.However, there are as yet no products in widespread clinical use.Existing methods, although extremely useful as research tools, lackadequate sensitivity for many practical applications, and are often toocomplicated to be carried out routinely and reliably in a clinicallaboratory.

In DNA therapeutics, an oligonucleotide is used to block the expressionof a specifically targeted gene by hybridizing to a complementarysequence within the corresponding mRNA and preventing the mRNA frombeing translated. In this manner, synthesis of the protein encoded bythe gene is blocked. Such is the basic concept employed in each of theabove three mentioned prior patents.

However, recent studies which formed the essence of the presentinvention have shown that blocking of the expression of the targetedgene occurs not simply by the hybridization of the oligonucleotide tothe mRNA, but requires that the RNA-DNA double helix so formed serve asa substrate for the enzyme RNaseH. RNaseH, a ubiquitous enzyme requiredfor DNA replication, then digests the mRNA at the RNA-DNA duplex. Onlythe mRNA is cleaved. The oligonucleotide is released intact and canreact repeatedly with other mRNA molecules as indicated by the dashedline in FIG. 1. The effect of each molecule of the oligonucleotide isthereby amplified, increasing the efficiency by which the expression ofthe targeted gene is blocked.

RNaseH acts only on RNA-DNA double helices. RNA-RNA duplexes do notserve as substrates for the enzyme. Therefore, oligodeoxyribonucleotides(short single-stranded DNA fragments) are preferred as therapeuticagents, rather than RNA molecules. Either RNA or DNA molecules can beused as probes for diagnostic purposes.

One problem faced in both DNA diagnostics and DNA therapeutics is thedegradation of the DNA or RNA molecule used either as a probe ortherapeutic agent by hydrolytic enzymes, termed nucleases, present inbody fluids and tissues. This problem is of far greater concern in thecase of DNA therapeutics. Oligonucleotides are degraded in blood, andeven more rapidly within cells, the rate of degradation beingsufficiently fast to completely abrogate the effect of the drug. Inpreparing clinical samples for DNA diagnostic tests, efforts are made toremove contaminating nucleases. However, low levels of activity mayremain. In certain instances, particularly when applied to genetictests, the detection method is based on specific cleavage of the probewhen it is hybridized to the target sequence. Non-specific degradationof the probe can give rise to an increased level of background signal,obscuring detection of the target-dependent cleavage of the probe.

Nucleases catalyze the degradation of DNA or RNA strands by hydrolysisof the phosphodiester bonds which join the nucleotide units within thechain, thereby cleaving the molecule into smaller fragments. DNA or RNAmolecules useful as hybridization probes range from approximately 15nucleotides in length to over several thousand nucleotides. DNAfragments useful as therapeutic agents range from about 10 to about 75nucleotides in length, preferably from 15 to 35 nucleotides. Thus, anyDNA or RNA molecule used as a probe or therapeutic agent would have manypotential sites at which cleaveage by nucleases may occur.

It is generally known in the field of molecular biology thatphosphodiester linkages normally subject to enzymatic cleavage bynucleases can be protected by modifying the phosphate group. Earlierefforts, such as the three mentioned patents, have focused onderivatives in which all of the phosphate groups within the strand aremodified, or on derivatives in which the phosphate groups areextensively modified in a random fashion following the synthesis of theoligonucleotide (Tullis, R. H., P.C.T. WO83/01451, 1983). Suchmodifications, however, often interfere with hybridization of the DNA toits target sequence, and consequently limits its utility for bothdiagnostic and therapeutic applications. Moreover, it has been foundthat DNA fragments in which the phosphate groups are extensivelymodified do not form substrates for RNaseH (Example 2). This abrogatestheir use as therapeutic agents.

While not wishing to be bound by any theory, the important discoveriesunderlying the present invention are: (1) that the mechanism by whicholigonucleotides used as therapeutic agents block the expression ofspecifically targeted genes is to hybridize to the corresponding mRNA,forming a substrate for RNaseH which then cleaves the mRNA; and (2) thatthe exclusive A pathway of degradation of oligonucleotides in blood, andthe predominant pathway within cells, is via sequential degradation fromthe 3′- to the 5′-end of the chain as outlined in FIG. 2. It has thusproven possible to greatly increase the stability of DNA fragments bymodification of only the first internucleotide link from the 3′-end ofthe DNA molecule. When this is done, cleavage of the first nucleotideunit is blocked, and no further degradation can proceed. A variety ofsubstitutions at the 3′-terminal internucleotide link, as explainedbelow, will serve this purpose, the position of the modification beingthe important factor. As demonstrated in Example 7, such modifiedderivatives have normal hybridization properties and do form substrateswith mRNAs that are recognized and cleaved by RNaseH. Such modifiedoligonucleotides, thus, are useful for both diagnostic and therapeuticapplications.

Oligonucleotides, although highly negatively charged molecules, do entercells at a finite rate. This was first suggested by a study carried outby Zamecnik and Stephenson (Proceedings of the National Academy ofSciences USA (1978) 75, 280-284 and 285-288), in which anoligonucleotide complementary to the 5′- and 3′-repeat sequences of Roussarcoma virus were found to inhibit viral replication and the synthesisof viral proteins in chick embryo fibroblasts. In these studies, themechanism of action of the oligonucleotide was not established, nor wasthe possiblity that the effects observed were due to degradationproducts of the oligonucleotide ruled out. Nonetheless, this study, withmore recent work, Heikkila et al., Nature (1987) 328, 445-449, indicatesthat oligonucleotides do enter cells. Zamecnik and Stephenson alsoshowed that the activity of the oligonucleotide could be substantiallyincreased by modifying the 3′-OH and 5′-OH groups with large apolarsubstituents, i.e., phenyl and naphthyl groups. These large apolargroups enhance the binding of the oligonucleotide to the cell membraneand transport into the cell. The introduction of these groups may alsohave inhibited the degradation of the oligonucleotide by exonucleases.However, only by directly modifying the phosphodiester group is itpossible to preclude cleavage of the internucleotide link within a DNAor RNA strand. Neither the degradation of the native oligonucleotide northe modified derivatives were studied in this work. Even the most recentstudy of the degradation of oligonucleotides is completely silent as tothe pathway by which degradation occurs (Wickstrom, E. (1986) Journal ofBiochemical and Biophysical Methods 13, 97-102).

Thus, while there has been some study of the degradation of DNA and RNAby endogenous nucleases within human and other mammalian tissuespreviously, it has not hithertofore been appreciated that degradation ofsingle-stranded DNA molecules occurs predominantly by a 3′- to5′-exonucleolytic mechanism. Hence the unique value of the modifiedderivatives claimed herein could not have been predicted. Put anotherway, because the pathway of degradation had not been established, nobasis existed for the design of DNA molecules in which only selectedphosphate groups within the chain were modified to effect an increase instability, such that the resulting oligonucleotide still hybridized tomRNAs to form substrates that are recognized and cleaved by RNaseH, asis required for use of the oligonucleotide in therapeutic applications.

As heretofore mentioned, the exact chemical modification at the3′-phosphodiester linkage is not as important as that the modificationbe at this precise location. A limited number of other positions on themolecule may also be modified without departing from the scope of thisinvention. In the case of DNA diagnostics, it is necessary to attach alabel onto the nucleic acid probe to enable its detection. Aradioactive, fluorescent, electrochemical, chemilumenescent or enzymemarker may serve this purpose. For oligonucleotide probes ranging inlength from 15 to about 35 nucleotides in length generally only onelabel is appended to the molecule. Longer DNA or RNA molecules used asprobes may be up to several thousand nucleotides in length. In this casethe label may be introduced at a density of up to 1 for every 15nucleotide units in the chain.

For applications in DNA therapeutics, the oligonucleotide may bemodified at positions in addition to the 3′-phosphodiester linkage toincrease its stability further or to facilitate its transport intocells. Transport of the oligonucleotide into the target cell may beenhanced by attachment to a carrier molecule normally taken up by thecell such as transferrin the plasma iron binding protein, or epidermalgrowth factor, or the oligonucleotide may be attached to an antibodyagainst a surface protein of the target cell as in the case of theso-called immunotoxins. Alternatively, additional phosphate groups ofthe oligonucleotide may be modified to reduce the negative charge on themolecule and thereby facilitate its transport across the lipid bilayerof the cell membrane. With such modifications, apolar substituents suchas hydrocarbon chains or aromatic groups may also be incorporated intothe molecule to further enhance intracellular uptake. Modification ofadditional phosphate groups within the chain may also increase thestability of the oligonucleotide further by blocking the activity of 5′-to 3′-exonucleases and endonucleases that are present in certain cells(see Example 4). Although degradation by these pathways is much lessrapid than degradation from the 3′-end of the chain, the added effect ofblocking these activities as well, particularly 5′- to 3′-exonucleolyticcleavage, may be beneficial in many cases. In these variousapplications, however, it is important that the modifications berestricted to a limited number of the phosphodiester groups. If most orall of the phosphodiester linkages within the chain are modified thiswill frequently interfere with the hybridization properties of theoligonucleotide, and more importantly, it will preclude the use of suchderivatives as therapeutic agents because they will no longer formsubstrates with targeted mRNAs that are acted upon by RNaseH. It isessential that there be one or more continuous stretches of theoligonucleotide chain in which the phosphodiester linkages areunmodified. The length of this unmodified portion of the chain must beat least 4 nucleotides, and preferably greater than 7 nucleotides inlength. When this is accomplished, the oligonucleotide will be protectedfrom degradation due to the modification of the 3′-terminalphosphodiester linkage, and will also hybridize with mRNAs to formsubstrates for RNaseH, which will cleave the mRNA across from theunmodified portion of the oligonucleotide, enabling the use of suchderivatives as therapeutic agents to block the expression of selectedgenes. The method of modifying the 3′-phosphodiester linkage will now bedescribed.

The modifying moiety is not critical. It can be selected from any of thefollowing functionalities:

In these structures, R may vary in length from 1 to about 20 carbonatoms, and may contain halogen substituents (as in Examples 5 and 6) orother heteroatoms, e.g., N, O, or S, attached to the chain. The methodsrequired to prepare these derivatives are simple, straight-forwardreactions well known to those skilled in the art. See for example, thefollowing literature references, each of which discloses modificationsof the type expressed herein: Letsinger, et al., Journal of the AmericanChemical Society, (1982), 104, 6805-6806; Stec et al., Journal of theAmerican Chemical Society (1984), 106, 6077-6079; Miller et al.,Biochemistry (1986), 25, 5092-5097; and Froehler, et al., TetrahedronLetters, (1986), 27, 469-472.

The preferred modification is a phosphotriester, for simplicity ofsynthesis and the diversity of functional groups which may be attachedto the phosphorus atom. Incorporation of a large hydrophobic substituentat this position, for example, may be utilized to facilitateintracellular uptake. As demonstrated in the present work,phosphotriesters are also resistant to hydrolysis by nucleases, and whenincorporated at only a limited number of positions within anoligonucleotide chain, the molecule remains able to hybridize normallywith mRNAs to form substrates which are cleaved by RNaseH.

In all methods now commonly used, oligonucleotides are synthesized fromthe 3′- to the 5′-end of the chain. The first residue is coupled to asolid support, such as controlled pore glass, through the 3′-OH group.If the synthesis is carried out in solution, rather than on a solidsupport, the 3′-OH of the first residue is blocked by a protecting groupwhich is removed at the culmination of the synthesis. Usually, onenucleotide unit is added at a time to the 5′-OH group of the growingchain. Alternatively, a block of several residues may be added in asingle reaction. There are several methods by which the internucleotidelink to the 5′-OH group may be formed. These procedures are reviewed inthe follwing monograph, “Oligonucleotide Synthesis: A PracticalApproach” (Gait, M. J., ed) IRL Press, Oxford (1984), which isincorporated herein by reference. The following reaction schemeillustrates the phosphoramidite method, the preferred chemistry atpresent.

DMT is the dimethoxytrityl protecting group; S.S. is the solid supporton which the synthesis is carried out. B₁ and B₂ are nucleic acid bases(either A, G, C or T).

The immediate product of this reaction is a phosphotriester. At the endof the synthesis, the protecting group R is removed to yield aphosphodiester linkage. Typically, the β-cyanoethyl group is used forthis purpose. Alternatively, R may be a group which is not removed, inwhich case the final product is a phosphotriester, the group R remainingpermanently attached to the phosphorus atom. It is in this manner that amodified phosphotriester may be incorporated at the firstinternucleotide linkage from the 3′-end of the chain. By essentially thesame chemistry an alkyl or aryl phosphonate may be introducedspecifically at this position, in which instance the group R is attacheddirectly to the phosphorous atom rather than through oxygen. Similarly,a hydrogen phosphonate may be introduced at this position. Suchderivatives may be used directly, or oxidized to give phosphotriestersor phosphoramidates incorporated at the 3′-terminal internucleotidelinkage. The following reference discloses modifications of the hydrogenphosphonate group, Froehler, B. C., Tetrahedron Letters, (1986), 27,5575-5578. A phosphorothioate or phosphoroselenate group may beincorporated at the 3′-terminal internucleotide link by carrying out theoxidation step in the phosphoramidite method with sulfur, or KSeCN,respectively, Stec. et al., Journal of the American Chemical Society,(1984), 106, 6077-6079. At the completion of the synthesis, followingthe removal of all protecting groups, the final product may be useddirectly or further purified by chromatographic methods well known tothose skilled in the art, see for example “Oligonucleotide Synthesis: APractical Approach” (Gait, M. J., ed.) IRL Press, Oxford (1984).

The procedures just described are generally applicable for the synthesisof oligonucleotides up to approximately 100 residues in length. In orderto synthesize a longer DNA or RNA molecule in which the 3′-terminalphosphodiester group is modified, as may be required for use as ahybridization probe in diagnostic tests, a combination of both chemicaland enzymatic methods is used. To accomplish the synthesis of such asequence, a short oligonucleotide in which the 3′-terminalphosphodiester linkage was modified, prepared by chemical methods, isenzymatically ligated onto the 3′-OH group of a longer DNA or RNA chain.

Methods of formulation and administration of the modifiedoligonucleotides described herein are obvious to those of ordinary skillin the medical arts, see for example, Avis, K. (1985) in Remington'sPharmaceutical Sciences, (Gennaro, A. R., ed) pp. 1518-1541, MackPublishing Company, Easton, Pa., which is incorporated herein byreference. Such methods of administration may include but are notlimited to topical, oral, or parenteral routes depending on the diseasestate. Appropriate vehicles for parenteral administration include 5%dextrose, normal saline, Ringer's solution and Ringer's lactate. Thematerial, of course, must be sterile and substantially endotoxin free.It may be possible in certain cases to store the product as alyophilized powder which would then be reconstituted when needed byaddition of an appropriate salt solution.

The following examples are offered to further illustrate, but not limit,the process, product and medical techniques of the invention, and serveto point out the unique features of the invention which enable it toovercome limitations of earlier contributions to the field.

EXAMPLE 1 Demonstration that the Inhibition of Translation of a TargetedmRNA by a Complementary Oligonucleotide is Dependent on the Cleavage ofthe mRNA by RNaseH

For the purpose of these experiments two oligonucleotides weresynthesized: 5′-GAGCACCATGGTTTC, MA-15; and5′-TGTCCAAGTGATTCAGGCCATCGTT,(SEQ ID NO:2) CODMB-25. Both were preparedon a Beckman automated DNA synthesizer using the phophoramidite method.MA-15 is complementary to a sequence within mouse α-globin mRNA spanningthe initiation codon. CODMB-25 hybridizes to a sequence within thecoding region of mouse β-globin mRNA. Mouse globin mRNA was isolatedfrom reticulocytes obtained from mice rendered anemic by treatment withphenylhydrazine as described by Goosens and Kan (Methods in Enzymology(Antonini, E., Rossi-Bernadi, L., and Chiancone, E., eds.) 76, 805-817,1981). In vitro translation reactions were carried out in the rabbitreticulocyte lysate system. The reticylocyte lysates used were purchasedfrom ProMega Biotec, and contain RNaseH. Translation reactions wereprogrammed with 1 ug of total mouse reticulocyte RNA. The reactions wereallowed to proceed for 1 hour at 30° C. Each sample contained 45 uCi of[³⁵S]methionine (Amersham) in a total volume of 25 ul. Protein synthesiswas measured by incorporation of [³⁵S]methionine into materialprecipitatable in 10% trichloracetic acid. In control reactions in theabsence of oligonucleotide, synthesis of β globin accounts for 60% ofthe incorporated counts; α globin synthesis accounts for 40% of theradioactivity incorporated into protein. Reactions were carried out inboth the presence and absence of poly rA/oligo dT (500 ug/ml). PolyrA/oligo dT is a competitive inhibitor of RNaseH, and at theconcentration used almost completely blocks the activity of the enzyme.Both MA-15 and CODMB-25 very effectively inhibited translation of thetargeted mRNA (see Table 1). MA-15 is complementary to 12 of 15 residuesin the corresponding sequence of β-globin mRNA, and, therefore, alsoinhibits β-globin synthesis to some extent. Poly rA/oligo dT completelyblocked the inhibitory effect of the oligonucleotides, indicating thatthe arrest of translation is mediated entirely by the cleavage of themRNA by RNaseH.

TABLE 1 Role of RNaseH in Hybrid-Arrested Translation % Inhibition OfGlobin Synthesis Oligonucleotide −poly rA/oligo dT +poly rA/oligo dTMA-15 (8M) 51 3 CODMB-25 (4 uM) 61 5

EXAMPLE 2 Oligodeoxynucleotide Methylphosphonates do not Form Substratesfor RNaseH

Two related substrates for RNaseH were compared: poly rA/oligo dT₁₂₋₁₈and poly rA/oligo dT₁₈ methylphosphonate (MP). Oligo dT₁₂₋₁₈ waspurchased from Pharmacia PL Biochemicals; the chain length varies from12 to 18 residues. Oligo dT₁₈MP was synthesized on a Beckman automatedDNA synthesizer using the phosphonamidite method. The 5′-terminalinternucleotide link is a normal phosphodiester; the remaining 16phosphate groups in the chain are methylphosphonates. Poly rA labeledwith tritium was purchased from Amersham.

Each reaction was conducted in a final volume of 100 ul containing 44picomoles of the substrate, either ³H-poly rA/oligo dT₁₂₋₁₈ or ³H-polyrA/oligo dT₁₈MP, in 50 mM Tris-HCl buffer pH 8.0, 20 mM KCl, 9 mM MgCl₂,1 mM 2-mercaptoethanol and 25 ug of bovine serum albumin. The reactionwas initiated by the addition of 2.3 units of E. coli RNaseH (BethesdaResearch Laboratories) and allowed to proceeed for 30 minutes at 37° C.After the reaction, 0.2 ml of carrier DNA solution (0.5 mg/ml of salmonsperm DNA, 0.1 M sodium pyrophosphate, and 1 mM ethylenediaminetetraacetic acid) plus 0.3 ml of 10% trichloracetic acid was added toeach sample. The tubes were then incubated on ice for 20 minutes, andafterwards centrifuged at 16,000 × g for 15 minutes. Cleavage of thepoly rA strand by RNaseH releases smaller fragments that are notprecipitated by trichloroacetic acid, and hence remain in thesupernatant. The supernatant was carefully removed, mixed with liquidscintillation cocktail, and counted for radioactivity. The reaction withpoly rA/oligo dT proceeded nearly to completion—85% of the radioactivecounts were solubilized. In contrast, less than 1% of poly rA/oligodT₁₈MP was digested by the enzyme. Thus, when much or all of thephosphate backbone of an oligodeoxynucleotide analogue is modified, itdoes not form a substrate with the complementary RNA sequence that isrecognized and cleaved by RNaseH.

EXAMPLE 3 Oligonucleotides are Degraded in Blood Exclusively by a 3′- to5′-Exonucleolytic Activity

The purpose of this study was to establish the pathways by whicholigonucleotides are degraded in blood. Surprisingly, a single pathwaywas observed: sequential degradation from the 3′- to the 5′-end of theDNA molecule.

The oligonucleotide MA-15 described in Example 1 was radioactivelylabeled with ³²P at the 5′-end of the molecule using γ³²P-adenosinetriphosphate (γ ³²P-ATP, Amersham) and T4 polynucleotide kinase(Bethesda Research Laboratories):

5-*GAGCACCATGGTTTC

where the asterisk represents the radiolabel. Separately, a radiolabeledderivative was prepared in which ³²P was introduced at the 3′mostinternucleotide link using α³²P-deoxycytidine triphosphate (dCTP) andDNA polymerase I (Bethesda Research Laboratories):

5′-GAGCACCATGGTTT*C

The reaction catalyzed by DNA polymerase I is between 5′-GAGCACCATGGTTTand dCTP and requires the complementary DNA strand as a template. A 50fold excess of unlabeled MA-15 was added after the reaction to insurethat the labeled derivative is single stranded. These procedures arestandard methods for radiolabeling DNA fragments, see for example,Maniatis, T., Fritsch, E. F., and Sambrook, J., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, pp. 114-115and 122-126, 1982, which is incorporated herein by reference.

The radiolabeled oligonucleotide 5×10⁵ counts/min) plus 2.5 nanomoles ofunlabeled MA-15 were added to 48 ul of freshly drawn human bloodanticoagulated with heparin. Samples were incubated at 37° C., bodytemperature. Aliquots of 6 ul were removed at 15 minutes, 30 minutes, 1hour, and 2 hours. A fraction of each aliquot was loaded onto apolyacrylamide gel and electrophoresed to separate the products on thebasis of size. To visualize the radiolabeled products, the gel wasexposed to x-ray film overnight. A drawing of the autoradiogram is shownin FIG. 3.

Degradation of the oligonucleotide labeled at the 5′-end of the molecule(lanes 1-5) gave rise to a ladder of discrete products of decreasingsize indicative of sequential exonucleolytic cleavage of the strand fromthe 3′-toward the 5′-end of the chain. Had cleavage occurred within themiddle of the strand, products of intermediate length would haveappeared throughout the time course. When MA-15 was labeled at the3′-end of the molecule, the resulting autoradiogram, lanes 6-10, showed,over time, disappearance of the full-length 15-mer, and a correspondingincrease in the mononucleotide pC. This result likewise demonstrates a3′- to 5′-exonucleolytic activity. The lack of products of intermediatesize rules out the presence of a 5′- to 3′-exonucleolytic activity, oran endonucleolytic (internal) cleavage activity.

By the same methods as just described, we showed that in blood fromother mammalian species, specifically mouse, rabbit, and rat,oligonucleotides are also degraded solely by sequential cleavage ofmononucleotides from the 3′-end of the chain.

EXAMPLE 4 The Principal Pathway of Degradation of Oligonucleotides inHuman Cells is by Sequential Exonucleolytic Cleavage of the Strand fromthe 3′- to the 5′-End of the Chain

The purpose of these studies was to determine the pathways by whicholigonucleotides are degraded by nucleases present within human cells.Radiolabeled derivatives of MA-15 were prepared as in Example 3.Degradation of the oligonucleotide was studied in extracts prepared fromthe human erythroleukemia cell line K562. The K562 cell line was firstisolated by Lozzio, C. B., and Lozzio, B. B., Blood (1975) 45, 321-334,and has been studied extensively as a model for red celldifferentiation. The cells were suspended in 10 mM tris buffer pH 7.4with 5 mM MgCl₂, 1 mM CaCl₂, 100 mM NaCl, and 0.1% Triton® X-100(polyethylene glycol 4-isooctylphenyl ether, and homogenized with a coldmotor driven teflon pestle homogenizer for 30 seconds. Cell membranesand organelles were further disrupted by sonication by five 15 secondbursts using a Biosonic sonifier set on full power. Remaining celldebris was removed from the extracts by centrifugation for 5 minutes at12,000×g.

The radiolabeled oligonucleotide was incubated in K562 cell extractsunder the same conditions as decribed in Example 3 and the degradationproducts analyzed by polyacrylamide gel electrophoresis. Degradation ofthe oligonucleotide was much faster than that in blood. The primarypathway of degradation was again a 3′ to 5′ exonucleolytic activity. Thehalf-life for cleavage of the 3′-terminal internucleotide link was 2minutes. A slower 5′- to 3′-exonucleolytic activity was also observed.The half-life for cleavage of the 5′-terminal nucleotide unit from thechain by this mechanism was 20 minutes.

Using the same methods as above, the pathways of degradation ofoligonucleotides in extracts prepared from other mammalian cells havealso been studied. These systems include African green monkey kidneycells (COS cells) and mouse liver cells. The results suggested a lowlevel of endonucleolytic activity (cleavage in the middle of thestrand), as well as a 5′- to 3′-exonuclease, but again the major pathwayof degradation of the oligonucleotide in each of these systems was by a3′- to 5′-exonuclease.

EXAMPLE 5 Modification of Oligonucleotides at the 3′-TerminalInternucleotide Link Completely Blocks Their Degradation in Human Blood

A derivative of the oligonucleotide MA-15 was prepared in which thefirst internucleotide link from the 3′-end of the molecule was modifiedas 2,2,2-trichloro-1,1-dimethylethyl phosphotriester. Thephosphotriester was introduced into the chain using the correspondingphosphorodichloridite:

To 20 micromoles of 5′-deoxythymidine dissolved in 1 cc of 10% pyridinein acetonitrile was added 19 micromoles of the phosphorodichlorodite.The reaction was allowed to proceed for 5 minutes at room temperature.The resulting monochlorodite was added to 1 micromole of deoxycytidineattached to controlled pore glass beads (American Bionetics). Thecoupling reaction to produce the dinucleotide phosphotriester wasallowed to proceed for 15 minutes at room temperature. Excess reagentsand by-products of the reaction were washed away with anhydrousacetonitrile. The remaining portion of the DNA strand was synthesized bythe β-cyanoethyl phosphoramidite method on a Beckman automated DNAsynthesizer. Only the 3′-terminal internucleotide link is modified. Theremaining 13 phosphate groups in the chain are normal phosphodiesters.

The modified oligonucleotide was labeled with ³²P at the 5′-terminal OHgroup using γ³²P-ATP and T4 polynucleotide kinase as described inExample 3. The radiolabeled oligonucleotide was incubated in human bloodat 37° C. Aliquots were removed at various times and analyzed bypolyacrylamide gel electrophoresis. A drawing of the autoradiogram ofthe gel is shown in FIG. 4. No degradation of the oligonucleotideoccurred over a period of 18 hours. The gradual decrease in theintensity of the band due to the full length molecule was due to removalof the ³²P label from the oligonucleotide by a phosphatase present inblood, not to cleavage of the strand.

Similar studies were carried out in which the modified oligonucleotidewas incubated in blood from the mouse. Again, no degradation of theoligonucleotide was observed.

EXAMPLE 6 Modification of Oligonucleotides at the 3′-TerminalInternucleotide Link Markedly Inhibits Their Degradation by NucleasesPresent in Human Cells

The derivative of MA-15 described in Example 5 in which the 3′-terminalinternucleotide link was modified as a 2,2,2-trichloro-1,1-dimethylethylphosphotriester was also used in this study. Extracts from K562 cellswere prepared as in Example 4. The modified oligonucleotide wasincubated in the cell extracts at 37° C. Aliquots were removed atvarious times, and the products of degradation were analyzed bypolyacrylamide gel electrophoresis.

Degradation of this modified oligonucleotide by the rapid 3′- to5′-exonuclease present in the cell extracts was completely blocked. Thisled to a marked increase in stability of the modified derivativecompared to the native oligonucleotide. The half-life was prolonged by10-fold. Degradation occurred by the slower 5′- to 3′-exonucleolyticactivity present in the K562 cell extracts (see Example 4). Thehalf-life for cleavage of the 5′-terminal nucleotide from the chain wasabout 20 minutes.

Similar studies were carried out in which the modified oligonucleotidewas incubated in extracts prepared from COS cells (African green monkeykidney) and mouse liver cells. In both cases, the half-life of thederivative modified at the 3′-terminal internucleotide link wasincreased 10- to 15-fold compared to the unmodified oligonucleotide.

EXAMPLE 7 Oligonucleotides Modified at the 3′-Terminal InternucleotideLinkage Form Substrates with mRNAs that are Cleaved by RNaseH

Mouse globin mRNA was isolated from mice rendered anemic by treatmentwith phenylhydrazine as described in Example 1. Three oligonucleotideswere compared in this study; MA-15, the derivative of MA-15 utilized inExamples 5 and 6 in which the 3′-terminal internucleotide link ismodified as a trichlorodimethylethyl phosphotriester (MA-15-TCDM), and aderivative further modified at the 5′-terminal OH group withnaphthylisocyanate (NPH-MA-15-TCDM). Mouse globin mRNA (2 micrograms)was added to 1.1 nanomoles of the oligonucleotide in a final volume of22 microliters containing 10 mM tris-HCl buffer at pH 7.5, 5 mM MgCl₂,25 mM NaCl, and 1 mM dithiothreitol. The reactions were initiated by theaddition of 2 units of E. coli RNaseH, and were allowed to proceed for30 minutes at 37° C. The reactions were stopped by extraction of thesamples with a 1:1 mixture of phenol and chloroform. The mRNA speciesremaining after digestion with RNaseH were isolated by precipitationwith ethanol and analyzed on Northern blots.

The RNA samples were reacted with 1 M glyoxal in a 1:1 (volume/volume)mixture of 10 mM sodium phosphate buffer at pH 6.5 and dimethylsulfoxidefor 1 hour at 50° C., and then electrophoresed on a 1.5% agarose gel.The RNAs were transferred from the gel to a Gene Screen Plus filter(DuPont-NEN) by capillary blotting according to the method described bythe manufacturer. Prehybridization of the filter was done in 1% sodiumdodecylsulfate, 1 M NaCl, 10% dextran sulfate, 50 mM sodium phosphate pH6.5 for 2 hours at 35° C. Hybridization was conducted in 5 ml of theprehybridization buffer containing 200 microgram/ml denatured salmonsperm DNA plus 2×10⁶ counts/min of MA-15, radiolabeled with ³²P at the5′-end of the molecule, for 24 hours at 35° C. After the hybridization,the filter was washed in the following order: once in 2×SSC (0.15 MNaCl, 0.015 M sodium citrate) for 5 minutes at room temperature, twicein 2×SSC plus 1% sodium dodecylsulfate for 30 minutes at 35° C., andonce in 0.1×SSC for 5 minutes at room temperature. The filter wasblotted dry and exposed to Kodak® XAR-5 film for autoradiography. Adrawing of the autoradiogram is shown in FIG. 5.

Using radiolabeled MA-15 as a probe, a single band due to α-globin mRNAwas detected on the Northern blot. In the absence of oligonucleotide, nocleavage of the mRNA occurred during the incubation with RNaseH (lane2). For the reaction in the presence of MA-15 (lane 3), a markeddecrease in the intensity of the band for α-globin mRNA is observed dueto the cleavage of the mRNA by RNaseH at the site of hybridization withthe probe. A similar decrease in intensity of the α-globin mRNA band wasobserved for the reactions with each of the modified oligonucleotides(lanes 4 and 5), indicating that they also hybridize with the mRNA toform a substrate that is recognized and cleaved by RNaseH.

EXAMPLE 8 Inhibition of Expression of the C-MYC Oncogene in the MurinePlasmacytoma Cell Line MOPC 315 with Complementary OligonucleotidesModified at the 3′-Terminal Internucleotide Link

C-myc is a cellular oncogene aberrantly expressed by a number of humanand animal tumor cells both in vivo and in cell culture. In the case ofthe murine plasmacytoma cell line MOPC 315, the c-myc gene istranslocated from its normal position on chromosome 15 to theimmunoglobulin locus on chromosome 12 and is expressed at an abnormallyhigh level.

Two oligonucleotide analogs (SEQ ID NO: 3) complementary to the sequenceof c-myc mRNA coding for amino acid residues 191 to 198 weresynthesized:

5′-G{circumflex over ( )}TAGGGAAAGACCACTGAGGGT{circumflex over ( )}C

5′-(PN)-GTAGGGAAAGACCACTGAGGGT{circumflex over ( )}C

where ({circumflex over ( )}) represents a trichlorodimethylethylphosphotriester and (PN)=

The TCDM phosphotriester was incorporated into the chain by theprocedure described in Example 5. The remaining portion of the sequencecontaining unmodified phosphodiester groups was synthesized by thephosphoramidite method. The 5′-terminal group (PN) was introduced usingthe reagent Aminolink from Applied Biosystems according to the proceduredescribed by the manufacturer. For comparison, the unmodifiedoligonucleotide having the same base sequence was also prepared, as wellas an oligonucleotide complementary to human albumin mRNA:

5′-T{circumflex over ( )}CCCTTCATCCCGAAGTT{circumflex over ( )}C (SEQ IDNO: 4)

where ({circumflex over ( )}) again represents the TCDM group.

MOPC 315 cells were maintained in RPMI 1640 culture media containing 10%fetal calf serum at 37° C. with 7% CO₂. Log phase MOPC 315 cells wereharvested by centrifugation (250×g for 19 minutes at 23° C.) andresuspended at a density of 5×10⁶/ml in HB101 serum-free medium(DuPont). One ml aliquots of this cell suspension were incubated in thepresence of oligonucleotide for 4 hours at 37° C. and 7% CO₂. Additionalone ml aliquots were incubated concommitantly without oligonucleotide.Cells from each group were then collected by centrifugation. Totalcellular RNA was prepared as described by Chirgwin et al., Biochemistry18, 5294-5299 (1979), and analyzed by Northern blots as described inExample 7. The filters were probed with an oligonucleotide complementaryto c-myc mRNA labeled with ³²P using polynucleotide kinase and γ³²P-ATPas in previous examples.

Both of the anti-c-myc oligonucleotides modified at the 3′-terminalinternucleotide link with the TCDM group, at a concentration of 1micromolar, decreased the level of c-myc mRNA by 75% when compared tothe steady-state level of the mRNA in MOPC 315 cells incubated in theabsence of oligonucleotide. The unmodified oligonucleotide against c-mycand the anti-albumin oligonucleotide had no effect on c-myc mRNA levelsat a concentration of either 1 or 5 micromolar. This result isconsistent with recent studies of another lymphoid cell line in which noinhibition of expression of the c-myc gene was found at concentrationsof an unmodified anti-c-myc oligonucleotide below a concentration of 15micromolar (Heikkila et al. (1987) Nature 328, 445-449). The modifiedderivatives described herein, thus, are between 20- and 50-fold moreeffective at inhibiting the expression of a targeted gene in intactcells than is the corresponding unmodified oligonucleotide.

In summary, the results presented show that oligonucleotides in whichthe 3′-terminal phosphodiester linkage is modified are resistant tonuclease digestion within cells and body fluids, retain normalhybridization properties with complementary nucleic acid sequences, andwhen hybridized to specifically targeted mRNA species form substrateswhich are recognized and cleaved by the enzyme RNaseH, enabling theoligonucleotide to be used to block the expression of the correspondinggene as is desired in various therapeutic applications.

Other modifications of the oligonucleotide may be made without departingfrom the scope and spirit of the invention, the important factor andcontribution being the discovery that modification of the 3′-terminalphosphodiester linkage alone is sufficient to markedly increase theresistance of the oligonucleotide to degradation, and that suchderivatives are able to form substrates with RNAs that are recognizedand cleaved by RNaseII. Additional substituents may be added to furtherincrease the stability of the oligonucleotide against degradation, tofacilitate intracellular transport, or to attach labels, e.g.,radioactive, fluorescent, or enzyme markers, for use of theoligonucleotide as a hybridization probe in diagnostic tests. It isimportant that such further modifications be restricted to a limitednumber of other sites on the molecule. If most or all of the phosphategroups within the chain are modified, the hybridization properties ofthe oligonucleotide may be adversely affected, and it will not formsubstrates with mRNAs that are acted upon by RNaseII. This latter factdefeats the use of highly modified oligonucleotide analogs astherapeutic agents to block the expression of specifically targetedgenes.

It therefore can be seen that the invention accomplishes at least all ofthe objectives heretofore stated.

5 15 base pairs nucleic acid single linear DNA unknown - /label= A/note= “May be labeled with radioactive phosphorous (P32) at the 5′-endof the molecule using gamma P32-adenosine triphosphate and a T4 - 15/label= B /note= ”May be labeled with radioactive phosphorous (P32) atthe 3′ most internucleotide link using an alpha P32-deoxycytidinetriphosphate 1 GAGCACCATG GTTTC 15 25 base pairs nucleic acid singlelinear DNA unknown 2 TGTCCAAGTG ATTCAGGCCA TCGTT 25 23 base pairsnucleic acid single linear DNA unknown - /label= A /note= “Between theguanine and thymine is a location where a tricholorodimethylethyl (TCDM)phosphotriester may be present.” - 23 /label= B /note= “Between thethymine and cytosine is a location where a trichlorodimethylethyl (TCDM)phosphotriester is present.” - /label= C /note= “Before the guanine,phosphoethyl amine (NH sub 2-CH sub 2-CH sub 2-O-PO sub 3 sup -) may beincorporated.” 3 GTAGGGAAAG ACCACTGAGG GTC 23 19 base pairs nucleic acidsingle linear DNA unknown - /label= A /note= “Between the thymine andcytosine, there exist a trichlorodimethylethyl phosphotriester.” - 19/label= B /note= “Between the thymine and cytosine, there exist atrichlorodimethylethyl phosphotriester.” 4 TCCCTTCATC CCGAAGTTC 19 10base pairs nucleic acid unknown unknown DNA unknown 5 ATGTCATTAC 10

What is claimed is:
 1. An oligodeoxynucleotide having: (a) a modified3′-terminal internucleotide phosphodiester linkage, which modified3′-terminal internucleotide phosphodiester linkage is selected from thegroup consisting of an alkyl or aryl phosphotriester, alkyl or arylphosphonate, hydrogen phosphonate, alkyl or aryl phosphoramidate orphosphoroselenate linkage and is resistant to 3′ to 5′ exonucleasedegradation, (b) one or more additional modified internucleotidephosphodiester linkages, which additional modified internucleotidephosphodiester linkage(s) is/are selected from the group consisting ofan alkyl or aryl phosphotriester, alkyl or aryl phosphonate, hydrogenphosphonate, alkyl or aryl phosphoramidate or phosphoroselenatelinkage(s) and is/are resistant to nuclease degradation, and (c) acontinuous stretch of at least five nucleotide residues having fourinternucleotide phosphodiester linkages which are unmodified, whereinsaid oligodeoxynucleotide, when mixed with an RNA molecule for which ithas complementarity under conditions in which an RNaseH is active,hybridizes to the RNA and forms a substrate that can be cleaved by theRNaseH.
 2. An oligodeoxynucleotide having: (a) a modified 3′-terminalinternucleotide phosphodiester linkage, which modified 3′-terminalinternucleotide phosphodiester linkage is resistant to 3′ to 5′exonuclease degradation, (b) one or more additional modifications, whichadditional modification(s) facilitate(s) intracellular transport of saidoligodeoxynucleotide, and (c) a continuous stretch of at least fivenucleotide residues having four internucleotide phosphodiester linkageswhich are unmodified, wherein said oligodeoxynucleotide, when mixed withan RNA molecule for which it has complementarity under conditions inwhich an RNaseH is active, hybridizes to the RNA and forms a substratethat can be cleaved by the RNAseH.
 3. The oligodeoxynucleotide of claim1 having one or more additional modifications, which additionalmodification(s) facilitate(s) intracellular transport of saidoligodeoxynucleotide.
 4. The oligodeoxynucleotide of claim 1 whereinsaid oligodeoxynucleotide is from about 10 to about 75 nucleotides inlength.
 5. The oligodeoxynucleotide of claim 1 wherein saidoligodeoxynucleotide is attached to a carrier molecule, which carriermolecule is taken up by a cell.
 6. The oligodeoxynucleotide of claim 1wherein the additional modified internucleotide phosphodiester linkageis at the 5′-terminal internucleotide phosphodiester linkage.
 7. Theoligodeoxynucleotide of claim 1 wherein the continuous stretch ofnucleotide residues having internucleotide phosphodiester linkages whichare unmodified is greater than eight nucleotide residues having greaterthan seven internucleotide phosphodiester linkages.
 8. Theoligonucleotide of claim 1 wherein all phosphodiester linkages outsidethe continuous stretch are modified.
 9. The oligodeoxynucleotide ofclaim 1 wherein the modified 3′-terminal internucleotide phosphodiesterlinkage is a phosphotriester linkage.
 10. The oligodeoxynucleotide ofclaim 1 wherein the modified 3′-terminal internucleotide phosphodiesterlinkage is a phosphonate linkage.
 11. The oligodeoxynucleotide of claim1 wherein the modified 3′-terminal internucleotide phosphodiesterlinkage is a phosphoramidate linkage.
 12. The oligodeoxynucleotide ofclaim 1 wherein the modified 3′-terminal internucleotide phosphodiesterlinkage is a phosphoroselenate linkage.
 13. The oligodeoxynucleotide ofclaim 1 wherein the additional modified internucleotide phosphodiesterlinkage(s) is/are a phosphotriester linkage(s).
 14. Theoligodeoxynucleotide of claim 1 wherein the additional modifiedinternucleotide phosphodiester linkage(s) is/are a phosphonatelinkage(s).
 15. The oligodeoxynucleotide of claim 1 wherein theadditional modified internucleotide phosphodiester linkage(s) is/are aphosphoramidate linkage(s).
 16. The oligodeoxynucleotide of claim 1wherein the additional modified internucleotide phosphodiesterlinkage(s) is/are a phosphoroselenate linkage(s).
 17. Anoligodeoxynucleotide having: (a) a phosphotriester linkage at the3′-terminal internucleotide position; (b) one or more additionalphosphotriester linkage(s) at the 5′ end of the oligodeoxynucleotide;and (c) a continuous stretch of at least five nucleotide residues havingfour internucleotide phosphodiester linkages which are unmodified. 18.An oligodeoxynucleotide having: (a) a phosphoramidate linkage at the3′-terminal internucleotide position; (b) one or more phosphoramidatelinkage(s) at the 5′ end of the oligodeoxynucleotide; and (c) acontinuous stretch of at least five nucleotide residues having fourinternucleotide phosphodiester linkages which are unmodified.
 19. Anoligodeoxynucleotide having: (a) a 3′-terminal internucleotidephosphodiester linkage, which modified phosphodiester linkage isresistant to 3′ to 5′ exonuclease activity, (b) an additionalmodification which comprises a label which permits detection of saidoligodeoxynucleotide; and (c) a continuous stretch of at least fivenucleotide residues having four internucleotide phosphodiester linkageswhich are unmodified, wherein said oligodeoxynucleotide, when mixed withan RNA molecule for which it has complementarity under conditions inwhich an RNaseH is active, hybridizes to the RNA and forms a substratethat can be cleaved by the RNAseH.
 20. An oligodeoxynucleotide having:(a) a modified 3′-terminal internucleotide phosphodiester linkage, whichmodified 3′-terminal internucleotide phosphodiester linkage is resistantto 3′ to 5′ exonuclease degradation, (b) a modified 5′-terminalinternucleotide phosphodiester linkage, which modified 5′-terminalinternucleotide phosphodiester linkage is resistant to nucleasedegradation, and (c) a continuous stretch of at least five nucleotideresidues having four internucleotide phosphodiester linkages which areunmodified, wherein said oligodeoxynucleotide, when mixed with an RNAmolecule for which it has complementarity under conditions in which anRNaseH is active, hybridizes to the RNA and forms a substrate that canbe cleaved by the RNaseH.
 21. An oligodeoxynucleotide having: (a) amodified 3′-terminal internucleotide phosphodiester linkage, whichmodified 3′-terminal internucleotide phosphodiester linkage is resistantto 3′ to 5′ exonuclease degradation, (b) a modified 5′-terminalinternucleotide phosphodiester linkage, which modified 5′-terminalinternucleotide phosphodiester linkage is resistant to nucleasedegradation, (c) one or more additional modifications, which additionalmodification(s) facilitate(s) intracellular transport of saidoligodeoxynucleotide, and (d) a continuous stretch of at least fivenucleotide residues having four internucleotide phosphodiester linkageswhich are unmodified, wherein said oligodeoxynucleotide, when mixed withan RNA molecule for which it has complementarity under conditions inwhich an RNAseH is active, hybridizes to the RNA and forms a substratethat can be cleaved by the RNaseH.
 22. The oligodeoxynucleotide of claim20 having one or more additional modifications, which additionalmodification(s) is/are resistant to nuclease degradation.
 23. Theoligodeoxynucleotide of claim 20 having one or more additionalmodifications, which additional modification(s) facilitate(s)intracellular transport of said oligodeoxynucleotide.
 24. Theoligodeoxynucleotide of claim 20 wherein said oligodeoxynucleotide isfrom about 10 to about 75 nucleotides in length.
 25. Theoligodeoxynucleotide of claim 20 wherein said oligodeoxynucleotide isattached to a carrier molecule, which carrier molecule is capable ofbeing taken up by a cell.
 26. The oligodeoxynucleotide of claim 20wherein the continuous stretch of nucleotide residues havinginternucleotide phosphodiester linkages which are unmodified is greaterthan eight nucleotide residues having greater than seven internucleotidephosphodiester linkages.
 27. The oligodeoxynucleotide of claim 20wherein all phosphodiester linkages outside the continuous stretch aremodified.
 28. The oligodeoxynucleotide of claim 20 wherein the modifiedinternucleotide linkages are phosphotriester, phosphonate,phosphoramidate, phosphorothioate or phosphoroselenate linkages.
 29. Anoligodeoxynucleotide having: (a) a modified 3′-terminal internucleotidephosphodiester linkage, which modified 3′-terminal internucleotidephosphodiester linkage is resistant to 3′ to 5′ exonuclease degradation,(b) at least one additional modified internucleotide phosphodiesterlinkage, which additional modified internucleotide phosphodiesterlinkage(s) is/are resistant to nuclease degradation, and (c) acontinuous stretch of at least five nucleotide residues having fourinternucleotide phosphodiester linkages which are unmodified, saidcontinuous stretch being flanked by modified internucleotidephosphodiester linkages resistant to nuclease degradation, wherein saidoligodeoxynucleotide, when mixed with an RNA molecule for which it hascomplementarity under conditions in which an RNaseH is active,hybridizes to the RNA and forms a substrate that can be cleaved by theRNaseH.
 30. The oligodeoxynucleotide of claim 29 having one or moreadditional modifications, which additional modification(s) facilitate(s)intracellular transport of said oligodeoxynucleotide.
 31. Theoligodeoxynucleotide of claim 2, 3, 21, 22, 23, or 30, wherein theadditional modification(s) is/are an additional modified internucleotidephosphodiester linkage(s).
 32. The oligodeoxynucleotide of claim 2, 3,21, 22, 23, or 30, wherein the additional modification(s) is anadditional modification of the 5′ terminal —OH group.
 33. Theoligodeoxynucleotide of claim 1, 22, and 30, wherein the additionalmodification(s) is/are a modified internucleotide phosphodiesterlinkage(s) selected from the group consisting of an alkyl or arylphosphotriester, alkyl or aryl phosphonate, hydrogen phosphonate, alkylor aryl phosphoramidate, phosphorothioate or phosphoroselenatelinkage(s).
 34. The oligodeoxynucleotide of claim 1, 2, 19, 20, 21, or29, wherein the RNaseH is E. coli RNaseH and the conditions in which itis active comprise a buffered solution of about pH 7.5 to about pH 8.0,about 5 mM to about 10 mM magnesium chloride and about 20 mM to about 25mM potassium chloride or sodium chloride, at 37° C.
 35. Theoligodeoxynucleotide of claim 1, 2, 3, 17, 18, 19, 20, 21, 22, 23, 29,or 30, wherein said oligodeoxynucleotide has complementarity for an mRNAof a transcribed gene.
 36. A composition comprising theoligodeoxynucleotide of claim 1, 2, 3, 17, 18, 19, 20, 21, 22, 23, 29,or 30, mixed with a carrier.